scholarly journals Evidence for the rapid direct control both in vivo and in vitro of the efficiency of oxidative phosphorylation by 3,5,3′-tri-iodo-l-thyronine in rats

1979 ◽  
Vol 184 (3) ◽  
pp. 527-538 ◽  
Author(s):  
R Palacios-Romero ◽  
J Mowbray
Keyword(s):  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Respiratory Control ◽  
Focused Attention ◽  
Isolated Mitochondria ◽  
Short Times ◽  
Normal Controls

1. Examination of the distribution of L-tri-iodothyronine among rat liver tissue fractions after its intravenous injection into thyroidectomized rats focused attention on mitochondria at very short times after administration. By 15 min this fraction contained 18.5% of the tissue pool; however, the content had decreased sharply by 60 min and even further over the next 3 h. By contrast, the content in all other fractions was constant or increased over 4 h. About 60% of tissue hormone was bound to soluble protein. 2. Mitochondria isolated from thyroidectomized rats showed P/O ratios that were about 50% of those found in normal controls, with both succinate and pyruvate plus malate as substrates. There was no evidence of uncoupling; the respiratory-control ratio was about 6. 3. Mitochondria isolated 15 min after injection of tri-iodothyronine into thyroidectomized rats showed P/O ratios and respiratory-control ratios that were indistinguishable from those obtained in mitochondria from euthyroid animals. The oxidation rate was, however, not restored. 4. Incubation of homogenates of livers taken from thyroidectomized animals injected with L-tri-iodothyronine before isolation of the mitochondria restored the P/O ratio to normal; by contrast, direct addition of hormone to isolated mitochondria had no effect. The role of extramitochondrial factors in rapid tri-iodothyronine action is discussed. 5. Possible mechanisms by which tri-iodothyronine might rapidly alter phosphorylation efficiency are considered: it is concluded that control of adenine nucleotide translocase is unlikely to be involved. 6. The amounts of adenine nucleotides in liver were measured both after thyroidectomy and 15 min after intravenous tri-iodo-thyronine administration to thyroidectomized animals. The concentrations found are consistent with a decreased phosphorylation efficiency in thyroidectomized animals. Tri-iodothyronine injection resulted in very significant changes in the amounts of ATP, ADP and AMP, and in the [ATP]/[ADP] ratio, consonant with those expected from an increased efficiency of ADP phosphorylation. This suggests that the changes seen in isolated mitochondria may indeed reflect a rapid response of liver in vivo to tri-iodo-thyronine.

scholarly journals Tissue-specific characterization of mitochondrial branched-chain keto acid oxidation using a multiplexed assay platform

2019 ◽  
Vol 476 (10) ◽  
pp. 1521-1537 ◽  
Author(s):  
Emma J. Goldberg ◽  
Katherine A. Buddo ◽  
Kelsey L. McLaughlin ◽  
Regina F. Fernandez ◽  
Andrea S. Pereyra ◽  
...  
Keyword(s):  
Acid Oxidation ◽  
Valuable Insight ◽  
Keto Acid ◽  
Isolated Mitochondria ◽  
Mouse Tissues ◽  
Branched Chain

Abstract Alterations to branched-chain keto acid (BCKA) oxidation have been implicated in a wide variety of human diseases, ranging from diabetes to cancer. Although global shifts in BCKA metabolism—evident by gene transcription, metabolite profiling, and in vivo flux analyses have been documented across various pathological conditions, the underlying biochemical mechanism(s) within the mitochondrion remain largely unknown. In vitro experiments using isolated mitochondria represent a powerful biochemical tool for elucidating the role of the mitochondrion in driving disease. Such analyses have routinely been utilized across disciplines to shed valuable insight into mitochondrial-linked pathologies. That said, few studies have attempted to model in vitro BCKA oxidation in isolated organelles. The impetus for the present study stemmed from the knowledge that complete oxidation of each of the three BCKAs involves a reaction dependent upon bicarbonate and ATP, both of which are not typically included in respiration experiments. Based on this, it was hypothesized that the inclusion of exogenous bicarbonate and stimulation of respiration using physiological shifts in ATP-free energy, rather than excess ADP, would allow for maximal BCKA-supported respiratory flux in isolated mitochondria. This hypothesis was confirmed in mitochondria from several mouse tissues, including heart, liver and skeletal muscle. What follows is a thorough characterization and validation of a novel biochemical tool for investigating BCKA metabolism in isolated mitochondria.

scholarly journals Highly efficient RNA-synthesizing system that uses isolated human mitochondria: new initiation events and in vivo-like processing patterns.

1984 ◽  
Vol 4 (8) ◽  
pp. 1605-1617 ◽  
Author(s):  
G Gaines ◽  
G Attardi
Keyword(s):  
Transcription Initiation ◽  
Ribosomal Proteins ◽  
High Efficiency ◽  
Rrna Synthesis ◽  
Isolated Mitochondria ◽  
Highly Efficient ◽  
Light Strand

A highly efficient RNA-synthesizing system with isolated HeLa cell mitochondria has been developed and characterized regarding its requirements and its products. In this system, transcription is initiated and the transcripts are processed in a way which closely reproduces the in vivo patterns. Total RNA labeling in isolated mitochondria proceeds at a constant rate for about 30 min at 37 degrees C; the estimated rate of synthesis is at least 10 to 15% of the in vivo rate. Polyadenylation of the mRNAs is less extensive in this system than in vivo. Furthermore, compared with the in vivo situation, rRNA synthesis in vitro is less efficient than mRNA synthesis. This is apparently due to a decreased rate of transcription initiation at the rRNA promoter and probably a tendency also for premature termination of the nascent rRNA chains. The 5'-end processing of rRNA also appears to be slowed down, and it is very sensitive to the incubation conditions, in contrast to mRNA processing. It is suggested that the lower efficiency and the lability of rRNA synthesis and processing in isolated mitochondria may be due to cessation of import from the cytoplasm of ribosomal proteins that play a crucial role in these processes. The formation of the light-strand-coded RNA 18 (7S RNA) is affected by high pH or high ATP concentration differently from the overall light-strand transcription. The dissociation of the two processes may have important implications for the mechanism of formation and the functional role of this unusual RNA species. The high efficiency, initiation capacity, and processing fidelity of the in vitro RNA-synthesizing system described here make it a valuable tool for the analysis of the role of nucleocytoplasmic-mitochondrial interactions in organelle gene expression.

Hepatotrophic effects of insulin on glucose, glycogen, and adenine nucleotides in hepatocytes isolated from fed adult rats

1980 ◽  
Vol 58 (10) ◽  
pp. 1004-1011 ◽  
Author(s):  
Khursheed N. Jeejeebhoy ◽  
Joseph Ho ◽  
Rajni Mehra ◽  
Alan Bruce-Robertson
Keyword(s):  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Energy Charge ◽  
Close Correlation ◽  
Adult Rats ◽  
Fed State ◽  
Adenine Nucleotide Pool ◽  
Intracellular Glucose

In vivo observations have suggested that there is an hepatotrophic effect of insulin. By contrast, subsequent in vitro work, using the isolated perfused liver system, showed no effect or indeterminate effects of insulin on the transport of glucose into the hepatocyte. However because this system may not have endured long enough to show such an influence we explored the transport of glucose using a 48-h suspension culture of hepatocytes isolated from young adult fed rats, the suspension being infused continuously with insulin at a rate approximating the maximum entering portal blood in the fed state. (In a separate study phloridzin was added after 2 h of incubation.) DNA, intracellular glucose and its inward transport, glycogen, and the adenine nucleotides were measured at intervals. By comparison with control or untreated cells, insulin-treated cells showed significantly more DNA and intracellular glucose, and the differences were abolished by phloridzin. Glucose transport rates fell to low values in untreated controls and still lower with insulin plus phloridzin. but the initial rate was maintained to the end (48 h) by insulin alone. Results for glycogen were similar to those for intracellular glucose. There was a close correlation (r = 0.96) between these two. The total adenine nucleotide pool and the concentration of ATP were maintained for about 24 h and fell to half their initial values by 48 h. Insulin had increased these concentrations significantly by 6 h. Although concentrations of ADP and AMP decreased gradually in all groups of cells, insulin enhanced the level of ADP by 12 h but had no measurable effect on that of AMP. The energy charge increased slightly throughout incubation but more so (by 6 h) in the presence of insulin. In conclusion the data support the concept that in the longer term (> 12 h) insulin in the portal circulation maintains the characteristic free permeability of the hepatocyte to glucose and this permits a variety of effects related to glucose entry into the hepatocyte.

scholarly journals Decrease in subunit aggregation of phosphoribosylpyrophosphate synthetase: a mechanism for decreased nucleotide concentrations in pyruvate kinase-deficient human erythrocytes

Blood ◽  
1986 ◽  
Vol 68 (5) ◽  
pp. 1024-1029
Author(s):  
CR Zerez ◽  
NA Lachant ◽  
KR Tanaka
Keyword(s):  
Pyruvate Kinase ◽  
Nicotinamide Adenine Dinucleotide ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Gel Permeation Chromatography ◽  
Metabolic Abnormalities ◽  
Gel Permeation ◽  
Reduced Nicotinamide Adenine Dinucleotide

Pyruvate kinase (PK)-deficient RBCs have several unexplained metabolic abnormalities, such as decreased concentrations of total adenine nucleotides (AMP, ADP, and ATP) and total (oxidized and reduced) nicotinamide adenine dinucleotide (NAD). Because 5-phosphoribosyl-1- pyrophosphate (PRPP) is an intermediate in the synthesis of adenine nucleotides and NAD, we investigated PRPP synthetase (PRPPS), the enzyme responsible for PRPP synthesis. This enzyme is regulated, in part, by changes in its state of subunit aggregation. The proportion of aggregated PRPPS can be altered in vitro by ATP and 2,3- diphosphoglycerate (DPG). Because PK-deficient RBCs have decreased ATP and increased DPG concentrations, we examined the state of subunit aggregation of PRPPS in RBCs from normal and PK-deficient subjects, using gel permeation chromatography. Young normal RBCs have more aggregated PRPPS than do older RBCs. In contrast, due to their decreased ATP and increased DPG concentrations, PK-deficient RBCs contain less aggregated PRPPS than do RBCs of comparable age without PK deficiency. These data suggest that PRPPS should be less active in vivo in PK-deficient RBCs. This may play a key role in mediating the decreases in total adenine nucleotide and total NAD concentrations in these RBCs.

scholarly journals Decrease in subunit aggregation of phosphoribosylpyrophosphate synthetase: a mechanism for decreased nucleotide concentrations in pyruvate kinase-deficient human erythrocytes

Blood ◽  
1986 ◽  
Vol 68 (5) ◽  
pp. 1024-1029 ◽  
Author(s):  
CR Zerez ◽  
NA Lachant ◽  
KR Tanaka
Keyword(s):  
Pyruvate Kinase ◽  
Nicotinamide Adenine Dinucleotide ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Gel Permeation Chromatography ◽  
Metabolic Abnormalities ◽  
Gel Permeation ◽  
Reduced Nicotinamide Adenine Dinucleotide

Abstract Pyruvate kinase (PK)-deficient RBCs have several unexplained metabolic abnormalities, such as decreased concentrations of total adenine nucleotides (AMP, ADP, and ATP) and total (oxidized and reduced) nicotinamide adenine dinucleotide (NAD). Because 5-phosphoribosyl-1- pyrophosphate (PRPP) is an intermediate in the synthesis of adenine nucleotides and NAD, we investigated PRPP synthetase (PRPPS), the enzyme responsible for PRPP synthesis. This enzyme is regulated, in part, by changes in its state of subunit aggregation. The proportion of aggregated PRPPS can be altered in vitro by ATP and 2,3- diphosphoglycerate (DPG). Because PK-deficient RBCs have decreased ATP and increased DPG concentrations, we examined the state of subunit aggregation of PRPPS in RBCs from normal and PK-deficient subjects, using gel permeation chromatography. Young normal RBCs have more aggregated PRPPS than do older RBCs. In contrast, due to their decreased ATP and increased DPG concentrations, PK-deficient RBCs contain less aggregated PRPPS than do RBCs of comparable age without PK deficiency. These data suggest that PRPPS should be less active in vivo in PK-deficient RBCs. This may play a key role in mediating the decreases in total adenine nucleotide and total NAD concentrations in these RBCs.

In vivo control of phosphofructokinase: system models suggest new experimental protocols

1989 ◽  
Vol 257 (4) ◽  
pp. R878-R888 ◽  
Author(s):  
R. J. Connett
Keyword(s):  
Adenine Nucleotide ◽  
Energy State ◽  
Adenine Nucleotides ◽  
Experimental Studies ◽  
Energy System ◽  
Absolute Size ◽  
Steady State Kinetics ◽  
Adenine Nucleotide Pool

There is still uncertainty as to how much control of in vivo rates of glycolysis by phosphofructokinase (PFK) depends on cytosolic phosphate energy state. Three models of PFK kinetics incorporating sensitivity to pH, adenine nucleotides, and inorganic phosphate (Pi) were embedded in the physiological "phosphate energy system" of creatine-containing tissues [Connett, R.J. Am. J. Physiol. 254 (Regulatory Integrative Comp. Physiol. 23): R949-R959, 1988]. Effects of changes in phosphate energy state and total adenine nucleotide and phosphate pools on steady-state kinetics were examined. Analyses mimicking in vitro experiments indicated no activity at the pH and [ATP] of working muscles. When tested using the coordinated changes in Pi and adenine nucleotides expected in vivo, all models showed reasonable activity. Control was dominated by [Pi] in the normal physiological range of energy states. The almost linear response to phosphate energy state, measured by creatine charge (phosphocreatine/total creatine), is insensitive to the absolute size of the adenine nucleotide pool. A step to almost full activation occurred when phosphocreatine buffering of [ATP] was exceeded. Several experimental studies are suggested.

Intracytoplasmic Concentration of GM-CSF and Intensity of the GM-CSF Membrane Receptor Are Significantly Higher in Proliferative Than in Dysplastic Variant of CMML and Could Explain the Higher In Vivo and In Vitro Growth Pattern.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2602-2602
Author(s):  
Francesco Onida ◽  
Servida Federica ◽  
Soligo Davide ◽  
Ricci Clara ◽  
Pasquini Maria Cristina ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Membrane Receptor ◽  
Cytokine Receptor ◽  
Biological Difference ◽  
Gm Csf ◽  
Significant Difference ◽  
Normal Controls

Abstract Chronic myelomonocytic leukemia (CMML) is a heterogeneous hematological malignancy, which has been included in a new category of MDS/MPD disorders in the last WHO classification of myeloid malignancies. An arbitrarily chosen leukocyte count has been proposed by the FAB group to differentiate between a “dysplastic” type (MD-CMML, with ≤12 x 109 WBC/L) and a “proliferative” type (MP-CMML, with >12 x 109 WBC/L) of CMML. However, apart from the WBC count, no biological difference has been identified to support distinction between these two disease-entities. Among factors that have been implicated in pathogenesis of CMML, GM-CSF produced by either autocrine or paracrine mechanisms has been shown to be a major growth determinant. In this study, peripheral blood samples from normal controls and from patients affected by proliferative and dysplastic variants of CMML were used to investigate expression of intracytoplasmic GM-CSF and expression of GM-CSF membrane receptor. Briefly, mononuclear cells (MNC) were isolated on Ficoll-Paque density gradient and cryopreserved in FCS 10% DMSO. In a first set of experiments samples from 5 healthy controls, 11 MP-CMML and 8 MD-CMML were thawed, permeabilized and stained with GM-CSF PE (Caltag) to evaluate expression of the intracytoplasmic cytokine by FACSCalibur flow cytometer (BD). Mean percentage of GM-CSF expression was 0.1 (range 0–0.5) in normal controls, 59.8 (range 14.5–90.7) in MP-CMML and 2.27 (range 0–9.3) in MD-CMML. The difference between MP and MD disease was statistically significant. To further investigate the possible role of GM-CSF cytokine in the pathogenesis of CMML, in a second set of experiments, MNC from 8 normal controls, 14 MP-CMML and 11 MD-CMML samples were thawed and stained with GM-CSFR (CD116 Pharmingen) and then with Goat Anti-Mouse FITC (BD) to evaluate the expression of the cytokine receptor. Mean percentage of expression of GM-CSFR was significantly higher in CMML samples (41.3, range 9.5–69) than in normal controls (20.3, range 16.4–27.3). No difference was detected between subtypes of MP-CMML and MD-CMML. When we considered median intensity of GM-CSFR expression, we observed a significantly higher values in MP-CMML than in MD-CMML (123.2 and 51.4, respectively), whereas no significant difference was detected between normal samples and MD-CMML. In this study, we also assessed "in vitro" spontaneous colony growth of PB-MNC from patients with both variants of CMML. The number of CFU-GM was higher in the MP-CMML than in MD-CMML (57 vs 17/5x10e5 cells plated) and a significant correlation with intracytoplasmic GM-CSF expression was observed (p <0.05). The higher levels of intracytoplasmic GM-CSF and the increased density of the cytokine receptor in MP-CMML suggest a possible role of GM-CSF in malignant cell proliferation of CMML patients. If our results will be confirmed, these findings could be utilized as a possible biological marker to distinguish proliferative and dysplastic variants of the disease. Further studies are warranted to investigate possible therapeutic applications.

scholarly journals Formate induces a metabolic switch in nucleotide and energy metabolism

2019 ◽  
Author(s):  
Kristell Oizel ◽  
Jacqueline Tait-Mulder ◽  
Jorge Fernandez-de-Cossio-Diaz ◽  
Matthias Pietzke ◽  
Holly Brunton ◽  
...  
Keyword(s):  
Energy Metabolism ◽  
De Novo ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Theoretical Modelling ◽  
Carbamoyl Phosphate ◽  
Metabolic Switch ◽  
Ampk Activity

Formate is a precursor for the de novo synthesis of purine and deoxythymidine nucleotides. Formate also interacts with energy metabolism by promoting the synthesis of adenine nucleotides. Here we use theoretical modelling together with metabolomics analysis to investigate the link between formate, nucleotide and energy metabolism. We uncover that endogenous or exogenous formate induces a metabolic switch from low to high adenine nucleotide levels, increasing the rate of glycolysis and repressing the AMPK activity. Formate also induces an increase in the pyrimidine precursor orotate and the urea cycle intermediate argininosuccinate, in agreement with the ATP dependent activities of carbamoyl-phosphate and argininosuccinate synthetase. In vivo data for mouse and human cancers confirms the association between increased formate production, nucleotide and energy metabolism. Finally, the in vitro observations are recapitulated in mice following intraperitoneal injection of formate. We conclude that formate is a potent regulator of purine, pyrimidine and energy metabolism.

scholarly journals GTP Is Required for Iron-Sulfur Cluster Biogenesis in Mitochondria

2007 ◽  
Vol 283 (3) ◽  
pp. 1362-1371 ◽  
Author(s):  
Boominathan Amutha ◽  
Donna M. Gordon ◽  
Yajuan Gu ◽  
Elise R. Lyver ◽  
Andrew Dancis ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Dependent Manner ◽  
Gtp Hydrolysis ◽  
Iron Sulfur Cluster ◽  
Isolated Mitochondria ◽  
Iron Sulfur ◽  
The Matrix

Iron-sulfur (Fe-S) cluster biogenesis in mitochondria is an essential process and is conserved from yeast to humans. Several proteins with Fe-S cluster cofactors reside in mitochondria, including aconitase [4Fe-4S] and ferredoxin [2Fe-2S]. We found that mitochondria isolated from wild-type yeast contain a pool of apoaconitase and machinery capable of forming new clusters and inserting them into this endogenous apoprotein pool. These observations allowed us to develop assays to assess the role of nucleotides (GTP and ATP) in cluster biogenesis in mitochondria. We show that Fe-S cluster biogenesis in isolated mitochondria is enhanced by the addition of GTP and ATP. Hydrolysis of both GTP and ATP is necessary, and the addition of ATP cannot circumvent processes that require GTP hydrolysis. Both in vivo and in vitro experiments suggest that GTP must enter into the matrix to exert its effects on cluster biogenesis. Upon import into isolated mitochondria, purified apoferredoxin can also be used as a substrate by the Fe-S cluster machinery in a GTP-dependent manner. GTP is likely required for a common step involved in the cluster biogenesis of aconitase and ferredoxin. To our knowledge this is the first report demonstrating a role of GTP in mitochondrial Fe-S cluster biogenesis.

scholarly journals Highly efficient RNA-synthesizing system that uses isolated human mitochondria: new initiation events and in vivo-like processing patterns

1984 ◽  
Vol 4 (8) ◽  
pp. 1605-1617
Author(s):  
G Gaines ◽  
G Attardi
Keyword(s):  
Transcription Initiation ◽  
Ribosomal Proteins ◽  
High Efficiency ◽  
Rrna Synthesis ◽  
Isolated Mitochondria ◽  
Highly Efficient ◽  
Light Strand

A highly efficient RNA-synthesizing system with isolated HeLa cell mitochondria has been developed and characterized regarding its requirements and its products. In this system, transcription is initiated and the transcripts are processed in a way which closely reproduces the in vivo patterns. Total RNA labeling in isolated mitochondria proceeds at a constant rate for about 30 min at 37 degrees C; the estimated rate of synthesis is at least 10 to 15% of the in vivo rate. Polyadenylation of the mRNAs is less extensive in this system than in vivo. Furthermore, compared with the in vivo situation, rRNA synthesis in vitro is less efficient than mRNA synthesis. This is apparently due to a decreased rate of transcription initiation at the rRNA promoter and probably a tendency also for premature termination of the nascent rRNA chains. The 5'-end processing of rRNA also appears to be slowed down, and it is very sensitive to the incubation conditions, in contrast to mRNA processing. It is suggested that the lower efficiency and the lability of rRNA synthesis and processing in isolated mitochondria may be due to cessation of import from the cytoplasm of ribosomal proteins that play a crucial role in these processes. The formation of the light-strand-coded RNA 18 (7S RNA) is affected by high pH or high ATP concentration differently from the overall light-strand transcription. The dissociation of the two processes may have important implications for the mechanism of formation and the functional role of this unusual RNA species. The high efficiency, initiation capacity, and processing fidelity of the in vitro RNA-synthesizing system described here make it a valuable tool for the analysis of the role of nucleocytoplasmic-mitochondrial interactions in organelle gene expression.

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